Facilitated urea transporters mediate urea movement across cellular plasma membranes. These proteins are the products of two closely related genes, UT-A and UT-B. We have characterised four mouse UT-A urea transporter homologues, mUT-A1, mUT-A2, mUT-A3 and mUT-A5. We have shown that an antibody raised in rabbit to the C-terminus of rat UT-A1 (L194) cross-reacts with mouse UT-As, because of the high degree of amino acid identity between rat and mouse UT-A1. Using L194 we have shown mUT-A1/2/4 are differentially expressed in several tissues, including kidney, brain, testes, liver and colon (Stewart et al. 2001). However, an antibody raised in rabbit to the C-terminus of rat UT-A3 does not cross-react with mouse UT-A3 because of low amino acid identity. We have therefore developed a novel antibody in chicken, termed MQ, raised to the C-terminal 15 amino acids of mouse UT-A3. In this study, we describe the characterisation of the MQ antibody.
Two chickens were immunised with the MQ peptide over a 3 month period and their eggs collected (Sigma Genosys). IgY was extracted from egg yolks using an Eggcellent IgY Purification Kit (Pierce, USA). The IgY fraction was then affinity purified using the original immunizing peptide and an Affi-gel binding column (Biorad, USA). Mouse tissue samples were obtained from humanely killed adult male MF1 mice. Western analysis with purified MQ identified proteins of 32, 45, 65 and 80 kDa differentially expressed in mouse kidney, heart, brain, testes, liver and colon. All signals were ablated by prior incubation with the original immunizing peptide. Deglycosylation of MQ immunoreactive proteins with PNGaseF enzyme caused the 32 kDa band to shift to 30 kDa, whereas the 45, 65 and 80 kDa bands were all unaffected. Interestingly, no immunoreactive bands were detected > 80 kDa, indicating that MQ did not recognise UT-A1. Immunolocalization in mouse kidney using MQ revealed immunostaining in collecting ducts in the middle and terminal renal inner medulla, in a pattern very similar to that previously described for UT-A3 expression in rat.
In conclusion, we have developed a novel polyclonal antibody raised to the C-terminal 15 amino acids of mouse UT-A3. This antibody preferentially recognises mouse UT-A3.
This work was funded by the BBSRC and the Royal Society.